TDP-43 Proteinopathy in Amyotrophic Lateral Sclerosis: Model, Mechanism, and Mediation

Amyotrophic lateral sclerosis (ALS) is the most common, fatal, and incurable motorneuron disease worldwide, characterized by degeneration of upper and lower motor neurons inthe central nervous system. Approximately 90% of ALS cases are sporadic with no hereditarybasis inheritance. Nevertheless, a unifying feature across both sporadic and familial forms ofALS, accounting for 97% of all ALS cases, is pathological aggregation of an essential nuclearregulatory protein—transactive response DNA binding protein (TDP-43). Several outstandingquestions regarding TDP-43 proteinopathy within the context of ALS remain: 1) How can TDP43 aggregates be modeled in vitro such that they recapitulate key features of aggregates foundin vivo such as co-aggregating proteins, sequestered genetic regulatory elements, and posttranslational modifications? 2) How does TDP-43 proteinopathy promote motor neurondegeneration through cell- and non-cell-autonomous mechanisms? And 3) what molecularstrategies can be implemented to mediate TDP-43 proteinopathy and restore its diverse andcritical functions as a global RNA regulator? In this work, I hypothesize that TDP-43 pathology exerts toxic gain-of-function effects via a non-cell-autonomous, neuroinflammatory mechanism. Further, this pathology provides an early-stage target for intervention using biological mediators such as molecular chaperones. This dissertation sought to address each of these three questions. In Chapter 2, Iintroduce a modified immunoprecipitation platform, coined TDiP, to model TDP-43 aggregatesakin to those in the ALS brain. Here, I used a series of biochemical, proteomics, and imagingstrategies and confirmed pathological similarity to aggregates identified in vivo; diseaseassociated post-translational modifications, nuclear factor sequestration, and microglial uptake.In Chapter 3, I implemented the TDiP protocol and established the foundation of a non-cell-autonomous mechanism of TDP-43-induced immune reactivity. Using a series of primaryimmune culture assays with subsequent validation in ALS patient tissue via spatialimmunophenotyping technology, I elucidated a potential antigenic property of TDP-43aggregates with the capacity to promote a maladaptive, innate-to-adaptive immune transition.Finally, in Chapter 4, I implemented genetic targeting and subcellular redistribution of amultimodal chaperone from the protein disulfide isomerase family and observed mediation ofseveral disease-associated phenotypes in vitro and in vivo. Altogether, this dissertation establishes new avenues for translational investigation intoTDP-43 proteinopathy as a pervasive pathological signature of ALS and several kindreddisorders.